The symbiotic relationship between leguminous plants and nitrogen-fixing bacteria involves nodule formation. Nodule formation is a complex process requiring communication between the bacteria and the host plant. During the initial events of symbiosis, plant-to-bacteria signal molecules known as flavonoids are produced by the host plant at low concentrations.
The flavonoids induce the expression of nod genes in the rhizobacteria bacteria. The first level of host specificity in the rhizobacteria-legume interaction is modulated by nodD and its alleles. These regulate the transcription of nodulation genes (nod, nol and noe). The nodD gene product, NodD, acts as a sensor of the plant signal and also regulates transcriptional regulation of the nod genes. The bacterial nod, nol and noe genes are required for infection and nodule formation.
Generally, the transcription of nol/noe genes is induced in the presence of NodD and flavonoids produced by host plants. NodD proteins belong to the LysR family of transcriptional activators. In the presence of flavonoids, NodD proteins bind to a nod box, a conserved promoter sequence preceding the inducible nod genes, and activate the transcription of nod operons.
Expression of the nod genes in the rhizobacteria is believed to be involved in the synthesis of lipo-chito oligosaccharides (LCOs). LCOs are substituted, β1,4 linked trimers, tetramers, and pentamers of N-acetylglucosamine. The LCOs, often described as “nod factors”, signal the plant and stimulate the formation of nodules inside the host plants.
Successful colonization of legume plants by nitrogen-fixing rhizobacteria is of significant agricultural and commercial importance. It would be particularly useful to obtain sources of LCOs that could be used to promote nodule formation by rhizobacteria. It would also be of benefit to identify compounds that are useful for inducing nod gene expression in rhizobacteria, resulting in production of LCOs. Rhizobacteria strains that are particularly responsive to nod gene induction, and which produce high levels of LCOs would also be of great utility.
A number of flavonoids which induce nod gene expression in rhizobacteria are known. Isoflavones, primarily genistein and diadzein, are the best inducers of nod::lacZ translational fusions and of the nod YABCUIJ operon in Bradyrhizobium japonicum. Genistein (C15H10O5, 5,7,4′-trihydroxyisoflavone, MW 270.2) is a stronger inducer of nod genes in B. japonicum than diadzein.
Jasmonic acid (JA) (Chemical Abstracts name: [1R-[1α,2β(Z)]]-3-oxo-2-(pentenyl)cyclopentaneacetic acid) and its methyl ester methyl jasmonate (MeJA), are fatty acid derived molecules. They are octadecanoid-based compounds that occur naturally in plants. Jasmonates are involved in plant growth and development, and play an important role in defence responses against pathogens and in wounding responses.
Jasmonic acid is produced in large quantities by the roots of wheat seedlings, and is also produced by fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. Jasmonic acid plays an important role in mycorrhizal signaling and, when applied to an ectomycorrhizal system, has been shown to increase the number of mycorrhized roots, and shoot and root dry weight of spruce seedlings.
Little is known with respect to how jasmonates affect the growth rate of symbiotic microorganisms, or the activation of bacteria-to-plant signaling molecules (nod factors) or their role in host-specific aspects of symbioses when they are present in the rhizosphere. Rosas et al (1998) recently reported that jasmonic acid and methyl jasmonate induced expression of nod genes in Rhizobium leguminasorum strain RBL 1284. However, Rosas et al. (1998) did not report whether jasmonic acid or methyl jasmonate increased LCO production as well.
The first step in jasmonic acid biosynthesis is the formation of linoleic acid (Chemical Abstracts name: (Z,Z)-9,12-Octadecadienoic acid) and linolenic acid (Chemical Abstracts name: (Z,Z,Z)-9,12,15-Octadecatrienoic acid) from membrane lipid breakdown, catalysed by phospholipase. Linoleic and linolenic acid are converted to 13-hydroperoxylinolenic acid by the action of lipoxygenase. 13-hydroperoxylinolenic acid is converted into 12,13 expoxy-octadecatrienoic acid in the presence of allene oxide synthase (AOS), and then converted into 12-oxo-phytodienoic acid by allene oxide cyclase (AOC). Following reduction and three steps of β-oxidation, (+)-7-iso-jasmonic acid is formed.
However, despite the role of linoleic and linolenic acid in the biosynthesis of jasmonic acid, it does not appear that they have been considered as possible inducers of nod gene expression or LCO production by rhizobacteria.
Not only is there a need for methods for increasing LCO production by rhizobacteria, there is a need for methods for increasing LCO production by rhizobacteria at low temperatures, in order to improve symbiotic nitrogen fixing symbiosis of rhizobacteria at low temperatures. Optimal symbiotic activity of rhizobacteria in legumes (i.e. nitrogen fixation) often occurs at a temperature far above that at which legume crops are grown. For instance, soybean is a subtropical legume that requires a root zone temperature (“RZT”) in the range of about 25 to 30° C. for optimal symbiotic activity. At low temperatures, expression of nod genes in B. japonicum, the soybean nitrogen fixing microsymbiont, are inhibited, resulting in a delayed onset of nodulation. Low spring soil temperature is therefore a major factor limiting soybean growth and symbiotic nitrogen fixation in northern regions, such as in Canada. Hence, methods for improving the symbiotic nitrogen fixing activity of rhizobacteria at low temperatures would be of great benefit to legume crop production in cool climates.